Researchers sit molecules for portraits in an ordered array of seats.

The (relatively) ancient art of crystallography has had a huge impact on modern science by showing us the location of atoms within a molecule. The structure of DNA (and how it acts as an information carrier) was worked out in part by using crystallography. The first step to understanding how a protein functions is often to figure out its structure using—you guessed it—crystallography. But despite its impact, crystallography feels a bit like an old-fashioned dark art.

To get a meaningful X-ray scattering pattern, the sample has to be crystallized in significant amounts. Without this, no structure can be determined (and crystallography really would be a dark art, in the sense of not producing any data). Crystallizing materials can be very difficult; obtaining crystals of rare, naturally occurring compounds is even more difficult. Much of X-ray crystallography is therefore devoted to coaxing recalcitrant molecules to align in regular patterns.

When a Japanese group of researchers reported X-ray crystallography without crystals, I couldn't resist delving in, even if we are a couple months late to the party.

Making X-rays work for you

X-ray diffraction patterns are formed by interference. The incoming X-rays penetrate deeply into a crystal, but a tiny fraction is scattered by each atom. Since a crystal has a regular array of atoms, the scattered X-rays add up in phase in some directions, resulting in a bright spot. By analyzing the bright spots, you can figure out the repeating pattern that scattered the X-rays.

This analysis is a bit more involved for complicated molecules, but the procedure is basically the same. Imagine that we replace each molecule with a single ball. These balls are repeated at regular intervals, which scatters the X-rays to create our pattern of bright spots. If we look a bit closer and we replace the balls with the original molecule, we see that every molecule has the same orientation, and each atom has a fixed spatial relationship to its corresponding atom in the next molecule over. This internal molecular structure creates its own diffraction pattern that overlays the basic pattern. Crystallographers spend a lot of time examining the details of which spots are present, how bright they are, and how a single spot might be broken up into multiple closely spaced spots, among other details.

The essential step is obtaining a sample of the material as a single crystal with sufficient size so that even the small spots are bright enough to see. This is often the limiting factor in X-ray diffraction analysis—getting around it is what makes this new technique so interesting.

Artificial crystals to the rescue

The ingenious step here was the recognition that it's the ordered structure that's important, not the crystal part. It just so happens that crystals are the most natural way to obtain order at the atomic and molecular scale.

But in recent years, scientists have figured out how to make crystal structures that have large, regular pores. These pores aren't just regularly arranged, they also have a regular internal structure—that is, the walls of each pore are identical to those in every other pore. This means that when a molecule diffuses into these open structures and sticks to the walls, it's going to adsorb to these surfaces in the same way and with the same orientation every time. If you have enough molecules sticking in enough pores, you create an ordered array of molecules.

The diffraction pattern is more complicated because it consists of all the contributions of the material that make up the walls of the pores and the molecule that is sitting in the pores. But because the support structure is known, it can be removed, leaving only the contribution due to the molecule of interest.

The researchers proved that this worked by performing a number of structural analyses of known molecules. They then took a molecule with a partially unknown structure and analyzed it to reveal the unknown details. In order to prove that the analysis wasn't a result of the researchers retrieving a structure they already knew should be there, they did the analysis blind. The researchers performing the structural analysis had no idea what they were analyzing.

An important aspect of this work is that the X-ray analysis requires much less raw material. The sample diffuses into the porous structure, meaning that it is very spread out compared to a normal crystal, so only a few micrograms are required (compared to milligrams for single crystal X-ray analysis). The point is that there are many purification techniques that manage to produce a few micrograms of material, but it's often very difficult and time-consuming to turn it into milligrams.

The true genius is realizing that only a single molecule per pore has to be in a fixed orientation. The rest can float around doing whatever they like. The disordered molecules simply add a smooth background to the entire pattern that is easily removed. In one case, the researchers estimated that only ten percent of the molecules that were present contributed to the X-ray pattern. This means that even when the binding between the pore surface and the sample molecule is not as specific as you would like, you're still likely to get reasonable data.

The downside is the size limitation. The molecules have to be able to diffuse into the pores, and the pores aren't that large. So at the moment, this is limited to small molecules. Unfortunately, the molecules we're most interested in learning about are proteins, which are the very opposite of small. Nevertheless, I wouldn't worry about that too much. I doubt we can use the same exact methods to make pores of a much larger diameter, but there are certainly other fabrication techniques that may provide two- and three-dimensional order arrays that allow individual protein molecules to artificially crystallize.

Chris Lee
Chris writes for Ars Technica's science section. A physicist by day and science writer by night, he specializes in quantum physics and optics. He lives and works in Eindhoven, the Netherlands. Emailchris.lee@arstechnica.com//Twitter@exMamaku

21 Reader Comments

Talk about voodoo arts... in our lab everyone generally spoke in a hushed voice when near the crystal-growing fridge. There was discussion of installing padded mats nearby (not seriously, at least I think not seriously).

I know, this is being picky, but the double helix structure of DNA was not proposed based on a crystal structure. DNA oligonucleotides suitable for crystallization were not available to Wilkins and Franklin. Instead, they used a random DNA sample in a slimy, globby state. By pulling the glob apart they formed a thin fiber of DNA connecting the two half globs in which the molecules were positioned more or less in parallel. This fiber was ordered enough to generate a diffraction pattern in an x-ray beam, and based on the pattern, some clever guesses and model building, Watson and Crick suggested the structure of the double helix. Real DNA crystallography didn't happen until the '80s, when Aaron Klug and others actually had access to DNA samples that were clean enough and homogeneous enough to grow crystals.

Very cool research, but for the most part, this field of work is foreign to me. What are purposes and implications of this type of research?

Figuring out what a molecule is shaped like is a very difficult problem. Since the shape of molecules is a major factor in how they behave in some types of reactions (ex organic catalysts) knowing what they look like is very valuable. Doing so computationally is enormously complex; which is why projects like folding@home are able to devour enormous amounts of donated computer time to get results that would otherwise be prohibitively expensive.

Xray crystallography is the semi-direct way to measure shape; this work potentially will make doing so much less difficult in the future. (Assuming it can be scaled up to larger molecules.)

Also, to me it's yet another idea that seems so obvious in retrospect. But if it really were so obvious, how come no one did it before? Is it because, in fact, it's not so obvious? Or is there some voodoo in building a perfect-enough substrate?

(Don't get me wrong, I'm entirely comfortable with the concept that some things seem obvious in hindsight, but only in hindsight.)

As an organometallic chemist I'd like to object to the claim that only large molecules are interesting structurally; There are lots of small molecule chemists who'd love to be able to get crystal structures of things we can't grow crystals of.

I wonder if using a specific type of ordered array might make it easier to visualize TM proteins? I've read that elucidating their structures with normal crystallographic techniques is notoriously difficult, since they usually have to be partly associated with a hydrophobic membrane to assume their native structures.

Also, to me it's yet another idea that seems so obvious in retrospect. But if it really were so obvious, how come no one did it before? Is it because, in fact, it's not so obvious? Or is there some voodoo in building a perfect-enough substrate?

(Don't get me wrong, I'm entirely comfortable with the concept that some things seem obvious in hindsight, but only in hindsight.)

Not only does this seem obvious, it also seems obvious that we should be able to use lithography to create a larger lattice combined with a DNA microarray to bind specifically to large proteins.

The non trivial part is to manufacture the microarray to a high degree of regularity, but it's even possible that the same techniques used in manufacturing CPUs can be used here, with a combination of etching, sputtering, and self-assembling support structures on top of the surface.

Of course then the issue is that self assembly is still in the research phases, so until that pans out you are left with lasers and acids and masks and old fashioned chemistry to attempt to create a nanostructure that can hold larger samples.

Very cool research, but for the most part, this field of work is foreign to me. What are purposes and implications of this type of research?

Figuring out what a molecule is shaped like is a very difficult problem. Since the shape of molecules is a major factor in how they behave in some types of reactions (ex organic catalysts) knowing what they look like is very valuable. Doing so computationally is enormously complex; which is why projects like folding@home are able to devour enormous amounts of donated computer time to get results that would otherwise be prohibitively expensive.

Xray crystallography is the semi-direct way to measure shape; this work potentially will make doing so much less difficult in the future. (Assuming it can be scaled up to larger molecules.)

There is also the issue that each protein has many possible conformations but worst part is that the most stable conformation (absolute minimum instead of just local minimum) is almost never the one that is active in the body. So even if the computer can calculate the secondary structure (it's 3D conformation) it can't really know which version is correct without actually simulating the production of the protein by the enzime as well as the surroundings during the production as they all influence the final secondary structure.

This does seem so simple in retrospect, but at the same time, I wasn't on the team that had to figure that one out.

I hate not having any institutional affiliation. It would almost be worth studying for the biochem. GRE just so I could have access to more than bloody abstracts without 5 hours of dedicated Google-fu.

How about an article series on the history of analytic techniques? Crystallography, spectrometry, chromatography, x-ray diffraction, all incredibly cool techniques, yet there seems to be a large discrepancy between knowledge of the practical aspects of their use and their historical underpinnings and development. I could spend the day studying up myself, but that would detract from time reading Ars!

This idea has been around for a while (at least 20 years or longer): to have proteins bind to a substrate matrix with known spacing to get around the phase problem. It is easier said than done. As others have already mentioned, this research applies more to small molecules than to proteins. But 1) there are still a lot of small molecules whose structures we want to know 2) the main promise of this method is LC-SCD analysis. I.e., if you have unknown substance(s) in solution, you determine what they are with X-ray single-crystal diffraction (not with mass spec or IR). You can use X-ray diffraction as analytical tool (e.g., in forensics); using nanogram amounts and getting your structure within hours. That's the revolutionary aspect of this paper and it just blows "classical" crystallographers' mind!

Do all molecules the right size to diffuse into the cavities bond and line up in a repeatable manner? Or do they have to contain specific functional groups to chemically interact? It would be odd if this were not the case.

Very cool research, but for the most part, this field of work is foreign to me. What are purposes and implications of this type of research?

Almost any technological development that depends on chemistry or material science benefits from actually knowing the structure of the chemicals or materials you're dealing with. There are various spectroscopic methods you can use to get structural information but in general, X-ray diffraction is the king, assuming you can get your stuff to crystallise.

For this particular research, I can see it being huge for studying catalytic systems, which are industrially important for cleaner, more efficient manufacture of just about every other chemical out there. There are two main types of catalytic system - heterogenous and homogenous. Heterogenous systems are where the phase of your catalyst (usually but not always a solid) is different to the phase of the stuff being catalysed. Think catalytic converters for vehicle exhaust systems. In homogenous systems, the catalyst and reactants are both in the same phase (usually liquid)

Homogenous systems are more flexible and can be a lot more selective and efficient but the big problem is separating out the catalyst at the end of your reaction. Step forward, immobilised catalysts, in which a homogenous catalyst is attached to a surface - say to the surface of a small bead. Theoretically this is the best of both worlds. Drop your beads into the reaction vessel, use your nice high selectivity catalyst to do whatever you need to do and then filter them out at the end. In practice, tethering the catalyst to a surface can change its structure in such a way that it no longer works.

The system described in the article sounds like an ideal way of studying the structure of small molecules attached to surfaces, hence an ideal tool for developing immobilised catalysts.

Also, to me it's yet another idea that seems so obvious in retrospect. But if it really were so obvious, how come no one did it before? Is it because, in fact, it's not so obvious? Or is there some voodoo in building a perfect-enough substrate?

(Don't get me wrong, I'm entirely comfortable with the concept that some things seem obvious in hindsight, but only in hindsight.)

My guess is that when it comes to diffraction patterns, subtracting a background pattern as described in the article, is not a trivial problem, particularly if you have a lot of background noise caused by 90% of your molecules that aren't sitting in an ordered array. For decent high resolution structures you need to collect diffraction data out to quite a large angle, which means measuring the intensity of all the small spots referred to in the article. Not easy with high background noise.

Just that it increases opportunities for understanding the relation between structure and properties. Favorite classical example: how metal atoms pack affects their ability to slip (or not, think fcc vs bcc) along planes, in directions of low resistance, which in turn determines mechanical properties. Of course that work didn't need the refinement described here, since those metals crystallize so completely. I also like the example of catalysts mentioned here in this thread: their structure plays a large role in their ability to facilatate chemical reactions.